Microparticle-drug conjugates for biological targeting

ABSTRACT

This invention provides novel methods and reagents for specifically delivering biologically active compounds to phagocytic mammalian cells. The invention also relates to specific uptake of such biologically active compounds by phagocytic cells and delivery of such compounds to specific sites intracellularly. The invention specifically relates to methods of facilitating the entry of antiviral and antimicrobial drugs and other agents into phagocytic cells and for targeting such compounds to specific organelles within the cell. The invention specifically provides compositions of matter and pharmaceutical embodiments of such compositions comprising conjugates of such antimicrobial drugs and agents covalently linked to particulate carriers generally termed microparticles. In particular embodiments, the antimicrobial drug is covalently linked to a microparticle via a cleavable linker moiety that is non-specifically cleaved in a phagocytic cell. In additional embodiments, the biologically-active compound is provided in an inactive, prodrug form that is activated by a chemical or enzymatic activity specific for cells infected by a microorganism, particularly a pathological or disease-causing microorganism. Thus, the invention provides cell targeting of drugs wherein the targeted drug is only activated in cells infected with a particular microorganism. Alternative embodiments of such specific drug delivery compositions also contain polar lipid carrier molecules effective in achieving intracellular organelle targeting in infected phagocytic mammalian cells. Particular embodiments of such conjugates comprise antimicrobial drugs covalently linked both to a microparticle via a cleavable linker molecule and to a polar lipid compound, to facilitate targeting of such drugs to particular subcellular organelles within the cell. Also provided are porous microparticles impregnated with antiviral and antimicrobial drugs and agents wherein the surface or outside extent of the microparticle is covered with a degradable coating that is degraded within a phagocytic mammalian cell. Also provided are nonporous microparticles coated with an antiviral or antimicrobial drug and further coated wherein the surface or outside extent of the microparticle is covered with a degradable coating that is degraded within a phagocytic mammalian cell. Methods of inhibiting, attenuating, arresting, combating and overcoming microbial infection of phagocytic mammalian cells in vivo and in vitro are also provided.

BACKGROUND OF THE INVENTION

This invention was made with government support under grant1-R01-CA49416 by the National Institutes of Health. The government hascertain rights in the invention.

1. Field of the Invention

This invention relates to methods of facilitating the entry ofbiologically-active compounds into phagocytic cells and for targetingsuch compounds to specific organelles within the cell. The inventionspecifically provides compositions of matter and pharmaceuticalembodiments of such compositions comprising conjugates of suchbiologically-active compounds covalently linked to particulate carriersgenerally termed microparticles. Particular embodiments of suchcompositions include compositions wherein the biologically-activecompounds are antiviral and antimicrobial drugs. In such compositionsthe microparticle is coated with an antiviral or antimicrobial drug, andthen further coated with organic coating material that is the target ofa microorganism-specific protein having enzymatic activity. Thus, theinvention provides cell targeting of drugs wherein the targeted drug isonly released in cells infected with a particular microorganism.Alternative embodiments of such specific drug delivery compositions alsocontain polar lipid carrier molecules. Particular embodiments of suchconjugates comprise a coated microparticle wherein an antiviral orantimicrobial drug is covalently linked to a polar lipid covalentlylinked to a polar lipid compound and the particle further coated with acoating material, to facilitate targeting of such drugs to particularsubcellular organelles within the cell.

2. Background of the Related Art

A major goal in the pharmacological arts has been the development ofmethods and compositions to facilitate the specific delivery oftherapeutic and other agents to the appropriate cells and tissues thatwould benefit from such treatment, and the avoidance of the generalphysiological effects of the inappropriate delivery of such agents toother cells or tissues of the body. The most common example of the needfor such specificity is in the field of antibiotic therapy, in which theamount of a variety of antibiotic, antiviral and antimicrobial agentsthat can be safely administered to a patient is limited by theircytotoxic and immunogenic effects.

It is also recognized in the medical arts that certain cells andsubcellular organelles are the sites of pharmacological action ofcertain drugs or are involved in the biological response to certainstimuli. In particular, it is now recognized that certain cell types andsubcellular organelles within such cell types are reservoirs for occultinfection that evades normal immune surveillance and permits thepersistence of chronic infections. Specific delivery of diagnostic ortherapeutic compounds to such intracellular organelles is thus desirableto increase the specificity and effectiveness of such clinicaldiagnostic or therapeutic techniques.

A. Drug Targeting

It is desirable to increase the efficiency and specificity ofadministration of a therapeutic agent to the cells of the relevanttissues in a variety of pathological states. This is particularlyimportant as relates to antiviral and antimicrobial drugs. These drugstypically have pleiotropic antibiotic and cytotoxic effects that damageor destroy uninfected cells as well as infected cells. Thus, anefficient delivery system which would enable the delivery of such drugsspecifically to infected cells would increase the efficacy of treatmentand reduce the associated “side effects” of such drug treatments, andalso serve to reduce morbidity and mortality associated with clinicaladministration of such drugs.

Numerous methods for enhancing the cytotoxic activity and thespecificity of antibiotic drug action have been proposed. One method,receptor targeting, involves linking the therapeutic agent to a ligandwhich has an affinity for a receptor expressed on the desired targetcell surface. Using this approach, an antimicrobial agent or drug isintended to adhere to the target cell following formation of aligand-receptor complex on the cell surface. Entry into the cell couldthen follow as the result of internalization of ligand-receptorcomplexes. Following internalization, the antimicrobial drug may thenexert its therapeutic effects directly on the cell.

One limitation of the receptor targeting approach lies in the fact thatthere are only a finite number of receptors on the surface of targetcells. It has been estimated that the maximum number of receptors on acell is approximately one million (Darnell et al., 1986, Molecular CellBiology, 2d ed., W.H. Freeman: New York, 1990). This estimate predictsthat there may be a maximum one million drug-conjugated ligand-receptorcomplexes on any given cell. Since not all of the ligand-receptorcomplexes may be internalized, and any given ligand-receptor system mayexpress many-fold fewer receptors on a given cell surface, the efficacyof intracellular drug delivery using this approach is uncertain. Otherknown intracellular ligand-receptor complexes (such as the steroidhormone receptor) express as few as ten thousand hormone molecules percell. Id. Thus, the ligand-receptor approach is plagued by a number ofbiological limitations.

Other methods of delivering therapeutic agents at concentrations higherthan those achievable through the receptor targeting process include theuse of lipid conjugates that have selective affinities for specificbiological membranes. These methods have met with little success. (see,for example, Remy et al., 1962, J. Org. Chem. 27: 2491-2500; Mukhergee &Heidelberger, 1962, Cancer Res. 22: 815-22; Brewster et al., 1985, J.Pharm. Sci. 77: 981-985).

Liposomes have also been used to attempt cell targeting. Rahman et al.,1982, Life Sci. 31: 2061-71 found that liposomes which containedgalactolipid as part of the lipid appeared to have a higher affinity forparenchymal cells than liposomes which lacked galactolipid. To date,however, efficient or specific drug delivery has not been predictablyachieved using drug-encapsulated liposomes. There remains a need for thedevelopment of cell-specific and organelle-specific targeting drugdelivery systems.

B. Phagocytic Cell-Specific Targeting

Cell-specific targeting is also an important goal of antimicrobialtherapy, particularly in the event that a specific cell type is a targetof acute or chronic infection. Targeting in the case of infection of aspecific cell type would be advantageous because it would allowadministration of biologically-toxic compounds to an animal sufferingfrom infection with a microbial pathogen, without the risk ofnon-specific toxicity to uninfected cells that would exist withnontargeted administration of the toxic compound. An additionaladvantage of such targeted antimicrobial therapy would be improvedpharmacokinetics that would result from specific concentration of theantimicrobial agent to the sites of infection, i.e., the infected cells.

Phagocytic cells such as monocytes and macrophages are known to bespecific targets for infection of certain pathogenic microorganisms.

Sturgill-Koszycki et al., 1994, Science 263: 678-681 disclose that thebasis for lack of acidification of phagosomes in M. avium and M.tuberculosis-infected macrophages is exclusion of the vesicularproton-ATPase.

Sierra-Honigman et al., 1993, J. Neuroiummunol. 45: 31-36 disclose Bornadisease virus infection of monocytic cells in bone marrow.

Maciejewski et al., 1993, Virol. 195: 327-336 disclose humancytomegalovirus infection of mononucleated phagocytes in vitro.

Alvarez-Dominguez et al., 1993, Infect. Immun. 61: 3664-3672 disclosethe involvement of complement factor C1q in phagocytosis of Listeriamonocytogenes by macrophages.

Kanno et al., 1993, J. Virol. 67: 2075-2082 disclose that Aleutian minkdisease parvovirus replication depends on differentiation state of theinfected macrophage.

Kanno et al., 1992, J. Virol. 66: 5305-5312 disclose that Aleutian minkdisease parvovirus infects peritoneal macrophages in mink.

Narayan et al., 1992, J. Rheumatol. 32: 25-32 disclose arthritis inanimals caused by infection of macrophage precursors with lentivirus,and activation of quiescent lentivirus infection upon differentiation ofsuch precursor cells into terminally-differentiated macrophages.

Horwitz, 1992, Curr. Top. Microbiol. Immunol. 181: 265-282 discloseLegionella pneumophila infections of alveolar macrophages as the basisfor Legionnaire's disease and Pontiac fever.

Sellon et al., 1992, J. Virol. 66: 5906-5913 disclose equine infectiousanemia virus replicates in tissue macrophages in vivo.

Groisman et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11939-11943disclose that S. typhimurium survives inside infected macrophages byresistance to antibacterial peptides.

Friedman et al., 1992, Infect. Immun. 60: 4578-4585 disclose Bordetellapertussis infection of human macrophages.

Stellrecht-Broomhall, 1991, Viral Immunol. 4: 269-280 disclose thatlymphocytic choriomeningitis virus infection of macrophages promotessevere anemia caused by macrophage phagocytosis of red blood cells.

Frehel et al., 1991, Infect. Immun. 59: 2207-2214 disclose infection ofspleen and liver-specific inflammatory macrophages by Mycobacteriumavium, the existence of the microbe in encapsulated phagosomes withinthe inflammatory macrophages and survival therein in phagolysosomes.

Bromberg et al., 1991, Infect. Immun. 59: 4715-4719 discloseintracellular infection of alveolar macrophages.

Mauel, 1990, J. Leukocyte Biol. 47: 187-193 disclose that Leishmaniaspp. are intracellular parasites in macrophages.

Buchmeier and Heffron, 1990, Science 248: 730-732 disclose thatSalmonella typhimurium infection of macrophages induced bacterial stressproteins.

Panuska et al., 1990, J. Clin. Invest. 86: 113-119 disclose productiveinfection of alveolar macrophages by respiratory syncytial virus.

Cordier et al., 1990, Clin. Immunol. Immunopathol. 55: 355-367 discloseinfection of alveolar macrophages by visna-maedi virus in chronicinterstitial lung disease in sheep.

Schlessinger and Horwitz, 1990, J. Clin. Invest. 85: 1304-1314 discloseMycobacterium leprae infection of macrophages.

Clarke et al., 1990, AIDS 4: 1133-1136 disclose human immunodeficiencyvirus infection of alveolar macrophages in lung.

Baroni et al., 1988, Am. J. Pathol. 133: 498-506 disclose humanimmunodeficiency virus infection of lymph nodes.

Payne et al, 1987, J. Exp. Med. 166: 1377-1389 disclose Mycobacteriumtuberculosis infection of macrophages.

Murray et al., 1987, J. Immunol. 138: 2290-2296 disclose that liverKupffer cells are the initial targets for L. donovani infection.

Koenig et al., 1986, Science 233: 1089-1093 disclose humanimmunodeficiency virus infection of macrophages in the central nervoussystem.

Horwitz and Maxfield, 1984, J. Cell Biol. 99: 1936-1943 disclose that L.pneumophila survives in infected phagocytic cells at least in part byinhibiting reduction of intraphagosomic hydrogen ion concentration (pH).

Shanley and Pesanti, 1983, Infect. Immunol. 41: 1352-1359 disclosecytomegalovirus infection of macrophages in murine cells.

Horwitz, 1983, J. Exp. Med. 158: 2108-2126 disclose that L. pneumophilais an obligate intracellular parasite that is phagocytized into aphagosome wherein fusion with lysosome is inhibited.

Chang, 1979, Exp. Parisitol. 48: 175-189 disclose Leischmania donovaniinfection of macrophages.

Wyrick and Brownridge, 1978, Infect. Immunol. 19: 1054-1060 discloseChlamydia psittaci infection of macrophages.

Nogueira and Cohn, 1976, J. Exp. Med. 143: 1402-1420 discloseTrypanosoma cruzi infection of macrophages.

Jones and Hirsch, 1972, J. Exp. Med. 136: 1173-1194 disclose Toxoplasmagondii infection of macrophages.

Persistent infection of phagocytic cells has been reported in the priorart.

Embretson et al., 1993, Nature 362: 359-361 disclose covert infection ofmacrophages with HIV and dissemination of infected cells throughout theimmune system early in the course of disease.

Schnorr et al., 1993, J. Virol. 67: 4760-4768 disclose measles viruspersistent infection in vitro in a human monocytic cell line.

Meltzer and Gendelman, 1992, Curr. Topics Microbiol. Immunol. 181:239-263 provide a review of HIV infection of tissue macrophages inbrain, liver, lung, skin, lymph nodes, and bone marrow, and involvementof macrophage infection in AIDS pathology.

Blight et al., 1992, Liver 12: 286-289 disclose persistent infection ofliver macrophages (Kuppfer cells) by hepatitis C virus.

McEntee et al., 1991, J. gen. Virol. 72: 317-324 disclose persistentinfection of macrophages by HIV resulting in destruction of Tlymphocytes by fusion with infected macrophages, and that themacrophages survive fusion to kill other T lymphocytes.

Kalter et al., 1991, J. Immunol. 146: 298-306 describe enhanced HIVreplication in macrophage CSF treated monocytes.

Meltzer et al., 1990, Immunol. Today 11: xx-yy describes HIV infectionof macrophages.

Kondo et al., 1991, J. gen. Virol. 72: 1401-1408 disclose herpes simplexvirus 6 latent infection of monocytes activated by differentiation intomacrophages.

King et al., 1990, J. Virol. 64: 5611-5616 disclose persistent infectionof macrophages with lymphocytic choriomeningitis virus.

Schmitt et al., 1990, Res. Virol. 141: 143-152 disclose a role for HIVinfection of Kupffer cells as reservoirs for HIV infection.

Gendelman et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7086-7090disclose lentiviral (visna-maedi) infection of bone marrow precursors ofperipheral blood monocytes/macrophages that provide a reservoir oflatently-infected cells.

Halstead et al., 1977, J. Exp. Med. 146: 201-217 disclose thatmacrophages are targets of persistent infection with dengue virus.

Mauel et al., 1973, Nature New Biol. 244: 93-94 disclose that lysis ofinfected macrophages with sodium dodecyl sulfate could release livemicrobes.

Attempts at drug targeting have been reported in the prior art.

Rubinstein et al., 1993, Pharm. Res. 10: 258-263 report colon targetingusing calcium pectinate (CaPec)-conjugated drugs, based on degradationof CaPec by colon specific (i.e., microflora-specific) enzymes and ahydrophobic drug incorporated into the insoluble CaPec matrices.

Sintov et al., 1993, Biomaterials 14: 483-490 report colon-specifictargeting using conjugation of drug to insoluble synthetic polymer usingdisaccharide cleaved by enzymes made by intestinal microflora,specifically, β-glycosidic linkages comprising dextran.

Franssen et al., 1992, J. Med. Chem. 35: 1246-1259 report renalcell/kidney drug targeting using low molecular weight proteins (LMWP) ascarriers, using enzymatic/chemical hydrolysis of a spacer moleculelinking the drug and LMWP carrier.

Bai et al., 1992, J. Pharm. Sci. 81: 113-116 report intestinal celltargeting using a peptide carrier-drug system wherein the conjugate iscleaved by an intestine-specific enzyme, prolidase.

Gaspar et al., 1992, Ann. Trop. Med. Parasitol. 86: 41-49 discloseprimaquine-loaded polyisohexylcyanoacrylate nanoparticles used to targetLeschmania donovani infected macrophage-like cells in vitro.

Pardridge, 1992, NIDA Res. Monograph 120: 153-168 reportopioid-conjugated chimeric peptide carriers for targeting to brainacross the blood-brain barrier.

Bai and Amidon, 1992, Pharm. Res. 9: 969-978 report peptide-drugconjugates for oral delivery and intestinal mucosal targeting of drugs.

Ashborn et al., 1991, J. Infect. Dis. 163: 703-709 disclose the use ofCD4-conjugated Pseudomonas aeruginosa exotoxin A to kill HIV-infectedmacrophages.

Larsen et al., 1991, Acta Pharm. Nord. 3: 41-44 report enzyme-mediatedrelease of drug from dextrin-drug conjugates by microflora-specificenzymes for colon targeting.

Faulk et al.,-1991, Biochem. Int. 25: 815-822 reportadriamycin-transferrin conjugates for tumor cell growth inhibition invitro.

Zhang and McCormick, 1991, Proc. Natl. Acad. Sci. USA 88: 10407-10410report renal cell targeting using vitamin B6-drug conjugates.

Blum et al., 1982, Int. J. Pharm. 12: 135-146 report polystyrenemicrospheres for specific delivery of compounds to liver and lung.

Trouet et al., 1982, Proc. Natl. Acad. Sci. USA 79: 626-629 report thatdaunorubicin-conjugated to proteins were cleaved by lysosomal hydrolasesin vivo and in vitro.

Shen et al., 1981, Biochem. Biophys. Res. Commun. 102: 1048-1052 reportpH-labile N-cis-acontinyl spacer moieties.

Monoclonal antibodies have been used in the prior art for drugtargeting.

Serino et al, U.S. Pat. No. 4,793,986, issued Dec. 27, 1988, providesplatinum anticancer drugs conjugated to polysaccharide (dextrin) carrierfor conjugation to monoclonal antibodies for tumor cell targeting.

Bickel et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2618-2622 disclosesthe use of a chimeric protein vector for targeting across blood-brainbarrier using anti-transferrin monoclonal antibody.

Rowlinson-Busza and Epenetos, 1992, Curr. Opin. Oncol. 4: 1142-1148provides antitumor immunotargeting using toxin-antibody conjugates.

Blakey, 1992, Acta Oncol. 31: 91-97 provides a review of antitumorantibody targeting of antineoplastic drugs.

Senter et al., 1991, in Immunobiology of Peptides and Proteins, Vol. VI,pp. 97-105 discloses monoclonal antibodies linked to alkalinephosphatase or penicillin-V amidase to activate prodrugs specifically atsite of antibody targeting, for therapeutic treatment of solid tumors.

Drug-carrier conjugates have been used in the prior art to providetime-release drug delivery agents,

Couveur and Puisieux, 1993, Adv. Drug Deliv. Rev. 10: 141-162 provide areview of microcapsule (vesicular), microsphere (dispersed matrix) andmicroparticle (1-250 μm)-based drug delivery systems, based ondegradation of particle with drug release, to provide time release ofdrugs, oral delivery via transit through the intestinal mucosa anddelivery to Kupffer cells of liver.

Duncan, 1992, Anticancer Drugs 3: 175-210 provide a review of improvedpharmicokinetic profile of in vivo drug release of anticancer drugsusing drug-polymer conjugates.

Heinrich et al., 1991, J. Pharm. Pharmacol. 43: 762-765 disclosepoly-lactide-glycolide polymers for slow release of gonadotropinreleasing hormone agonists as injectable implants.

Wada et al. 1991, J. Pharm. Pharmacol. 43: 605-608 disclosesustained-release drug conjugates with lactic acid oligomers.

Specifically, polymer-conjugated drugs have been reported in the priorart, and attempts to adapt particulate conjugates have also beenreported.

Ryser et al., U.S. Pat. No. 4,847,240, issued Jul. 11, 1989, providescationic polymers for conjugation to compounds that are poorlytransported into cells. Examples include the antineoplastic drugmethotrexate conjugated with polylysine and other polycationic aminoacids are the carriers.

Ellestad et al., U.S. Pat. No. 5,053,394, issued Oct. 1, 1991, providescarrier-drug conjugates of methyltrithiol antibacterial and antitumoragents with a spacer linked to a targeting molecule which is an antibodyor fragment thereof, growth factors or steroids.

Kopecek et al., U.S. Pat. No. 5,258,453, issued Nov. 2, 1993, providesantitumor compositions comprising both an anticancer drug and aphotoactivatable drug attached to a copolymeric carrier by functionalgroups labile in cellular lysosomes, optionally containing a targetingmoiety that are monoclonal antibodies, hormones, etc.

Negre et al., 1992, Antimicrob. Agents and Chemother. 36: 2228-2232disclose the use of neutral mannose-substituted polylysine conjugateswith an anti-leischmanial drug (allopurinol riboside) to treat murineinfected macrophages in vitro.

Yatvin, 1991, Select. Cancer. Therapeut. 7: 23-28 discusses the use ofparticulate carriers for drug targeting.

Hunter et al., 1988, J. Pharm. Phamacol. 40: 161-165 discloseliposome-mediated delivery of anti-leischmanial drugs to infected murinemacrophages in vitro.

Saffran et al., 1986, Science 233: 1081-1084 disclose drug release froma particulate carrier in the gut resulting from degradation of thecarrier by enzymes produced by intestinal microflora.

Targeting of specific dyes and localization of the components of certainpathological organisms to the Golgi apparatus has been reported in theprior art.

Lipsky & Pagano, 1985, Science 228: 745-747 describe Golgi-specificvital dyes.

Pagano & Sleight, 1985, Science 229: 1051-1057 describes lipid transportin mammalian cells.

Pagano et al., 1989, J. Cell Biol. 109: 2067-2079 describes localizationof fluorescent ceramide derivatives to the Golgi apparatus.

Barklis & Yatvin, 1992, Membrane Interactions of HIV, Wiley-Liss: N.Y.,pp. 215-236 describe membrane organization of HIV viral coat in infectedmammalian cells.

SUMMARY OF THE INVENTION

The present invention is directed to an improved method for deliveringbiologically-active compounds to phagocytic cells and cellularorganelles of such phagocytic cells in vivo and in vitro. This deliverysystem achieves such specific delivery of biologically-active compoundsin inactive, prodrug form which are then specifically activated within aphagocytic cell, most preferably a phagocytic mammalian cell, infectedwith a microorganism, most preferably a pathological or disease-causingmicroorganism. In preferred embodiments, the inactive prodrugs of theinvention are provided as conjugates between polar lipids andbiologically-active compounds. In one preferred embodiment of theinvention is provided a biologically-active compound in inactive,prodrug form that can be delivered to phagocytic cells throughconjugating the compound with a microparticle via an cleavable linkermoiety. Alternatively, specific delivery is achieved by impregnating abiologically-active compound in inactive, prodrug form into a porousmicroparticle which is then coated with a coating material. In analternative embodiment, the delivery system comprises a nonporousmicroparticle wherein a biologically-active compound in inactive,prodrug form is made to coat the particle, and the particle is thenfurther coated by a coating material. As used herein, these differentembodiments of the microparticles of the invention are genericallydefined as “microparticle-conjugated” embodiments. In preferredembodiments of each aspect of the invention, the biologically-activecompound in inactive, prodrug form is most preferably provided as aconjugate of the biologically-active compound with a polar lipid via aspecific linker moiety that is specifically cleaved in a phagocyticcell, most preferably a phagocytic mammalian cell, infected with amicroorganism, most preferably a pathological or disease-causingmicroorganism, wherein the inactivated prodrug form of thebiologically-active compound is activated thereby.

In each case, non-specific release of the polarlipid-biologically-active compound conjugate is achieved by enzymatic orchemical release of the inactive, prodrug form of thebiologically-active compound from the microparticle by cleavage of thecleavable linker moiety or the coating material in a phagocytic cells,followed by specific release of the biologically-active compound inparticular phagocytic cells. In preferred embodiments, wherein thebiologically-active compound in inactive, prodrug form is provided as aconjugate of the biologically-active compound with a polar lipid via aspecific linker moiety, activation is specifically accomplished bychemical or enzymatic cleavage of a specific linker moiety between thebiologically-active compound and the polar lipid. Most preferably, thebiologically-active compound is inactive or has reduced activity in theform of a polar lipid conjugate, wherein the activity of the compound isrestored or increased upon specific cleavage of the linker moiety in aparticular phagocytic cell. In preferred embodiments, the specificlinker moiety is enzymatically cleaved by an enzyme that is produced bya microorganism, most preferably a pathological or disease-causingmicroorganism or which is induced by infection by a microorganism, mostpreferably a pathological or disease-causing microorganism. Inadditional preferred embodiments, the specific linker moiety ischemically cleaved under physiological conditions that are specific forphagocytic cells infected with a microorganism, most preferably apathological or disease-causing microorganism.

In addition, conjugation of the biologically-active compound with apolar lipid provides for targeting of the conjugate to specificsubcellular organelles. This invention has the specific advantage offacilitating the delivery of such compounds to specific subcellularorganelles via the polar lipid carrier, achieving effectiveintracellular concentrations of such compounds more efficiently and withmore specificity than conventional delivery systems. Moreover, thetargeted biologically-active compounds comprising the conjugates arespecifically activated or their activity increased at the intracellulartarget by cleavage of the specific linker moiety and release of thebiologically-active compound at the targeted intracellular site.

The specific delivery of biologically-active compounds to phagocyticcells, most preferably phagocytic mammalian cell, is achieved by thepresent invention by chemical or physical association of the inactiveprodrug form of the biologically-active compounds with a microparticle.Specific intracellular accumulation and facilitated cell entry ismediated by the phagocytic uptake of microparticle-conjugatedbiologically active compounds by such cells. Preferred embodiments ofphagocytic cellular targets include phagocytic hematopoietic cells,preferably macrophages and phagocytic neutrophils.

Particularly preferred targets of the microparticle-conjugatedbiologically active compounds of the invention are phagocytic cells,preferably macrophages and phagocytic neutrophils, and in particularsuch cells that are infected with any of a variety of microorganism,most preferably a pathological or disease-causing microorganism. Forsuch cells, the embodiments of the microparticle-conjugated biologicallyactive compounds of the invention are comprised of cleavable linkermoieties whereby chemical or enzymatic cleavage of said linker moietiesare non-specifically cleaved inside the cells, and in preferredembodiments, inside phagocytic cells, wherein specific activation of theinactive prodrug to the active form of the biologically-active compoundis achieved specifically in infected cells. In preferred embodiments,the inactive prodrugs are provided as conjugates of thebiologically-active compounds with a polar lipid moiety via a specificlinker moiety, wherein the biologically-active compound is activatedfrom the prodrug state in phagocytic cells infected with amicroorganism, most preferably a pathological or disease-causingmicroorganism, via specific cleavage of the linker moiety forming theconjugate between the polar lipid and the biologically-active compound.This provides for the specific release of biologically-active compounds,such as antiviral and antimicrobial drugs, to such infected cells,preferably targeted to specific intracellular targets for more effectivedelivery of such drugs within an infected phagocytic cell. It isunderstood that all phagocytic cells will take up such antiviral andantimicrobial embodiments of the microparticle-conjugated biologicallyactive compounds of the invention, and will cleave the cleavable linkerso as to release the prodrug form of the biologically-active compound inall phagocytic cells. However, it is an advantageous feature of themicroparticle-conjugated biologically active compounds of the inventionthat specific activation of the inactive prodrug form of thebiologically-active compounds is achieved only in phagocytic cellsinfected with a microorganism, most preferably a pathological ordisease-causing microorganism. Release of biologically-active forms ofsuch antiviral and antimicrobial drugs is dependent on the presence ofthe infectious microorganism in the phagocytic cell, in preferredembodiments, by cleavage of the specific linker moiety comprising thepolar lipid-biologically active compound conjugate.

In preferred embodiments of this aspect of the invention, thebiologically active compounds of the invention linked to microparticlesvia the cleavable linker are covalently linked to a polar lipid moiety.Polar lipid moieties comprise one or a plurality of polar lipidmolecules. Polar lipid conjugates of the invention are comprised of oneor a plurality of polar lipid molecules covalently linked to abiologically-active compound via a specific linker moiety as describedabove. Such specific linker moieties are provided having two linkerfunctional groups, wherein the linker has a first end and a second endand wherein the polar lipid moiety is attached to the first end of thelinker through a first linker functional group and thebiologically-active compound is attached to the second end of the linkerthrough a second linker functional group. In these embodiments of theinvention, the linker functional groups attached to the first end andsecond ends of the linker are characterized as “strong”, with referenceto the propensity of the covalent bonds between each end of the linkermolecule to be broken. In preferred embodiments of this aspect of theinvention, the propensity of the covalent bonds between each of the endsof the linker molecule is low, that is, the polar lipid/biologicallyactive compound conjugate is stable under intracellular physiologicalconditions in the absence of a chemical or enzymatic moiety specific forcellular infection by a microorganism, most preferably a pathological ordisease-causing microorganism. In these embodiments, the specific linkermoiety allows the biologically-active compound to accumulate and act atan intracellular site after being released from the microparticle onlyafter having been released from the intracellular targeting polar lipidmoiety.

In a particular embodiment of this aspect of the invention, the specificlinker moiety is a peptide of formula (amino acid)_(n), wherein n is aninteger between 2 and 100, preferably wherein the peptide comprises apolymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, thebiologically-active compound of the invention has a first functionallinker group, and a polar lipid moiety has a second functional linkergroup, and the compound is directly covalently linked to the polar lipidmoiety by a chemical bond between the first and second functional linkergroups. In such embodiments, either the biologically-active compound orthe polar lipid moiety comprises yet another functional linker groupwhich is directly covalently linked to the cleavable linker moiety ofthe invention, which in turn is covalently linked to the microparticle.In preferred embodiments, each of the functional linker groups is ahydroxyl group, a primary or secondary amino group, a phosphate group orsubstituted derivatives thereof or a carboxylic acid group. Inparticular, in such embodiments the polar lipid/biologically activecompound conjugate is preferably specifically cleaved in infectedphagocytic mammalian cells. In these embodiments, thebiologically-active compound is an inactive, prodrug state whencovalently linked to the polar lipid, which activity of the biologicallyactive compound in restored or increased after the conjugate has beenbroken.

In the various aspects of the polar lipid conjugates of the invention,preferred polar lipids include but are not limited to acyl carnitine,acylated carnitine, sphingosine, ceramide, phosphatidyl choline,phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol,phosphatidyl serine, cardiolipin and phosphatidic acid.

Preferred biologically active compounds comprising the polar lipidconjugates linked to the microparticles of the invention includeantiviral and antimicrobial compounds, drugs, peptides, toxins and otherantibiotic agents.

The invention also provides compositions of matter comprising a porousmicroparticle into which is impregnated an inactive, prodrug form of abiologically-active compound, the impregnated porous microparticle beingfurther coated with a coating material. In this aspect of the invention,the coating material is non-specifically degraded by chemical orenzymatic means inside a cell, preferably a phagocytic mammalian cell,allowing the release of the in inactive, prodrug form of the compoundfrom the microparticle. In preferred embodiments, the coating materialis a substrate for a protein having an enzymatic activity found cells,preferably mammalian phagocytic cells. In additional preferredembodiments, the biologically-active compound in inactive, prodrug formis provided as a conjugate of the biologically-active compound with apolar lipid via a specific linker moiety. In such embodiments,activation of the inactive prodrug is specifically accomplished bychemical or enzymatic cleavage of a specific linker moiety between thebiologically-active compound and the polar lipid. Most preferably, thebiologically-active compound is inactive or has reduced activity in theform of a polar lipid conjugate, wherein the activity of the compound isrestored or increased upon specific cleavage of the linker moiety in aparticular phagocytic cell that is infected with a microorganism,preferably a pathological or disease-causing microorganism.

In preferred embodiments, specific release of the biologically-activecompound in particular phagocytic cells that are infected with amicroorganism, most preferably a pathological or disease-causingmicroorganism is achieved via specific cleavage of a specific linkermoiety that forms the conjugate between the polar lipid and thebiologically-active compound. In these preferred embodiments, cleavageof the specific linker moiety is achieved by chemical or enzymaticcleavage of the linker moiety between the biologically-active compoundand the polar lipid. Preferably, the biologically-active compound isinactive or has reduced activity in the form of a polar lipid conjugate,wherein the activity of the compound is restored or increased uponspecific cleavage of the linker moiety in a particular phagocytic cell.In preferred embodiments, the specific linker moiety is enzymaticallycleaved by an enzyme that is produced by a microorganism, mostpreferably a pathological or disease-causing microorganism or which isinduced by infection by a microorganism, most preferably a pathologicalor disease-causing microorganism. In additional preferred embodiments,the specific linker moiety is chemically cleaved under physiologicalconditions that are specific for phagocytic cells infected with amicroorganism, most preferably a pathological or disease-causingmicroorganism.

Preferred biologically active compounds comprising the polar lipidconjugates used to impregnate such porous microparticles includeantiviral and antimicrobial compounds, drugs, peptides, toxins and otherantibiotic agents.

In these embodiments, the biologically active compounds of the inventionimpregnated within porous microparticles are covalently linked to apolar lipid moiety. Polar lipid moieties comprise one or a plurality ofpolar lipid molecules. Polar lipid conjugates of the invention arecomprised of one or a plurality of polar lipid molecules covalentlylinked to a biologically-active compound via a specific linker moiety asdescribed above. Such specific linker moieties are provided having twolinker functional groups, wherein the linker has a first end and asecond end and wherein the polar lipid moiety is attached to the firstend of the linker through a first linker functional group and thebiologically-active compound is attached to the second end of the linkerthrough a second linker functional group. In these embodiments of theinvention, the linker functional groups attached to the first end andsecond ends of the linker is characterized as “strong”, with referenceto the propensity of the covalent bonds between each end of the linkermolecule to be broken. In these embodiments, the specific linker moietyallows the biologically-active compound to accumulate and act at anintracellular site after being released from the microparticle onlyafter having been released from the intracellular targeting polar lipidmoiety. In these embodiments, the propensity of the covalent bondsbetween each of the ends of the linker molecule is low, that is, thepolar lipid/biologically active compound conjugate is stable underintracellular physiological conditions in the absence of a chemical orenzymatic moiety specific for cellular infection by a microorganism,most preferably a pathological or disease-causing microorganism.

In a particular embodiment of this aspect of the invention, the specificlinker moiety is a peptide of formula (amino acid)_(n), wherein n is aninteger between 2 and 100, preferably wherein the peptide comprises apolymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, thebiologically-active compound of the invention has a first functionallinker group, and a polar lipid moiety has a second functional linkergroup, and the compound is directly covalently linked to the polar lipidmoiety by a chemical bond between the first and second functional linkergroups. In such embodiments, either the biologically-active compound orthe polar lipid moiety comprises yet another functional linker groupwhich is directly covalently linked to the cleavable linker moiety ofthe invention, which in turn is covalently linked to the microparticle.In preferred embodiments, each of the functional linker groups is ahydroxyl group, a primary or secondary amino group, a phosphate group orsubstituted derivatives thereof or a carboxylic acid group. Inparticular, in such embodiments the polar lipid/biologically activecompound conjugate is preferably specifically cleaved in infectedphagocytic mammalian cells, wherein the activity of the biologicallyactive compound in restored or increased after the conjugate has beenbroken.

In the various aspects of the polar lipid conjugates of the invention,preferred polar lipids include but are not limited to acyl carnitine,acylated carnitine, sphingosine, ceramide, phosphatidyl choline,phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol,phosphatidyl serine, cardiolipin and phosphatidic acid.

The invention also provides compositions of matter comprising anonporous microparticle onto which is coated a an inactive, prodrug formof a biologically-active compound, the non-porous microparticle beingfurther coated with a coating material. In this aspect of the invention,the coating material is non-specifically degraded inside a cell,preferably a phagocytic mammalian cell, allowing the release of theinactive, prodrug form of the compound from the microparticle. Inpreferred embodiments, the coating material is a substrate for a proteinhaving an enzymatic activity found cells, preferably mammalianphagocytic cells. In additional preferred embodiments, thebiologically-active compound in inactive, prodrug form is provided as aconjugate of the biologically-active compound with a polar lipid via aspecific linker moiety. In such embodiments, activation of the inactiveprodrug is specifically accomplished by chemical or enzymatic cleavageof a specific linker moiety between the biologically-active compound andthe polar lipid. Most preferably, the biologically-active compound isinactive or has reduced activity in the form of a polar lipid conjugate,wherein the activity of the compound is restored or increased uponspecific cleavage of the linker moiety in a particular phagocytic cellthat is infected with a microorganism, preferably a pathological ordisease-musing microorganism.

In preferred embodiments, specific release of the biologically-activecompound in particular phagocytic cells that are infected with amicroorganism, most preferably a pathological or disease-causingmicroorganism is achieved via specific cleavage of the linker moietyforming the conjugate between the polar lipid and thebiologically-active compound. In preferred embodiments, cleavage of thespecific linker moiety is achieved by chemical or enzymatic cleavage ofthe linker moiety between the biologically-active compound and the polarlipid. Preferably, the biologically-active compound is inactive or hasreduced activity in the form of a polar lipid conjugate, wherein theactivity of the compound is restored or increased upon specific cleavageof the linker moiety in a particular phagocytic cell. In preferredembodiments, the specific linker moiety is enzymatically cleaved by anenzyme that is produced by a microorganism, most preferably apathological or disease-causing microorganism or which is induced byinfection by a microorganism, most preferably a pathological ordisease-causing microorganism. In additional preferred embodiments, thespecific linker moiety is chemically cleaved under physiologicalconditions that are specific for phagocytic cells infected with amicroorganism, most preferably a pathological or disease-causingmicroorganism.

In this aspect of the invention, the inactive, prodrug form of thebiologically-active compound of the invention will be understood todissolve from the surface of the microparticle upon enzymatic orchemical degradation of the coating material. Release of thebiologically-active compound can be accomplished simply be mass action,i.e., whereby the compound dissolves from the surface of the nonporousmicroparticle into the surrounding cytoplasm within the cell.

Preferred biologically active compounds used to prepare the coated,non-porous microparticles of this aspect of the invention includeantiviral and antimicrobial compounds, drugs, peptides, toxins and otherantibiotic agents.

In preferred embodiments, the biologically active compounds of theinvention coated onto nonporous microparticles are covalently linked toa polar lipid moiety. Polar lipid moieties comprise one or a pluralityof polar lipid molecules. The polar lipid conjugates of the inventionare comprised of one or a plurality of polar lipid molecules covalentlylinked to a biologically-active compound via a specific linker moiety asdescribed above. Such specific linker moieties are provided having twolinker functional groups, wherein the linker has a first end and asecond end and wherein the polar lipid moiety is attached to the firstend of the linker through a first linker functional group and thebiologically-active compound is attached to the second end of the linkerthrough a second linker functional group. In these embodiments of theinvention, the linker functional groups attached to the first end andsecond ends of the linker is characterized as “strong”, with referenceto the propensity of the covalent bonds between each end of the linkermolecule to be broken. In preferred embodiments of this aspect of theinvention, the specific linker moiety allows the biologically-activecompound to act at an intracellular site after being released from themicroparticle only after having been released from the intracellulartargeting polar lipid moiety. In these embodiments, the propensity ofthe covalent bonds between each of the ends of the linker molecule islow, that is, the polar lipid/biologically active compound conjugate isstable under intracellular physiological conditions in the absence of achemical or enzymatic moiety specific for cellular infection by amicroorganism, most preferably a pathological or disease-causingmicroorganism.

In a particular embodiment of this aspect of the invention, the specificlinker moiety is a peptide of formula (amino acid)_(n), wherein n is aninteger between 2 and 100, preferably wherein the peptide comprises apolymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, thebiologically-active compound of the invention has a first functionallinker group, and a polar lipid moiety has a second functional linkergroup, and the compound is directly covalently linked to the polar lipidmoiety by a chemical bond between the first and second functional linkergroups. In such embodiments the polar lipid/biologically-activeconjugate is the inactive, prodrug form of the biologically-activecompound. Said conjugate is impregnated into porous microparticles orcoats non-porous microparticles as described above. In preferredembodiments, each of the functional linker groups is a hydroxyl group, aprimary or secondary amino group, a phosphate group or substitutedderivatives thereof or a carboxylic acid group. In particular, in suchembodiments the polar lipid/biologically active compound conjugate ispreferably specifically cleaved in infected phagocytic mammalian cells.In these embodiments, the biologically-active compound is an inactive,prodrug state when covalently linked to the polar lipid, which activityof the biologically active compound in restored or increased after theconjugate has been broken.

In the various aspects of the polar lipid conjugates of the invention,preferred polar lipids include but are not limited to acyl carnitine,acylated carnitine, sphingosine, ceramide, phosphatidyl choline,phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol,phosphatidyl serine, cardiolipin and phosphatidic acid.

The invention also provides compositions of matter comprising abiologically-active compound in an inactive, prodrug form, wherein theprodrug is linked to a microparticle via a cleavable linker moiety. Thecleavable linker moieties of the invention comprise two linkerfunctional groups, wherein the cleavable linker moiety has a first endand a second end. The microparticle is attached to the first end of thecleavable linker moiety through a first linker functional group and theinactive, prodrug form of the biologically-active compound is attachedto the second end of the cleavable linker moiety through a second linkerfunctional group. The cleavable linker moieties of the invention arenon-specifically cleaved inside a cell, preferably a phagocyticmammalian cell. In this aspect of the microparticles of the invention,the biologically active compound is provided in a non-biologicallyactive form, wherein the compound is not activated merely by releasefrom the microparticle. Rather, in this aspect of the microparticles ofthe invention, the biologically-active compound is specificallyactivated in a cell, preferably a phagocytic mammalian cell, that isinfected with a microorganism, most preferably a pathological ordisease-causing microorganism. In preferred embodiments, thebiologically active compound is specifically activated by an enzymaticactivity produced by a microorganism, most preferably a pathological ordisease-musing microorganism or which is induced by infection by amicroorganism, most preferably a pathological or disease-causingmicroorganism. In additional preferred embodiments, the biologicallyactive compound is specifically activated by a chemical reaction underphysiological conditions that are specific for phagocytic cells infectedwith a microorganism, most preferably a pathological or disease-causingmicroorganism.

In this aspect of the invention are also provided embodiments whereinthe biologically active compound is covalently linked to a polar lipidmoiety. Polar lipid moieties comprise one or a plurality of polar lipidmolecules. Polar lipid conjugates of the invention are comprised of oneor a plurality of polar lipid molecules covalently linked to abiologically-active compound. In preferred embodiments, activation ofthe biologically active compound as described above is achieved byspecific cleavage of a covalent bond between the biologically activecompound and a polar lipid moiety, or by specific cleavage of a specificlinker moiety that comprises the conjugate between the polar lipid andthe biologically-active compound.

In preferred embodiments of the invention, the biologically-activecompound is a peptide. In other preferred embodiments, thebiologically-active compound is a drug, most preferably an antiviral orantimicrobial drug. Preferred polar lipids include but are not limitedto acyl carnitine, acylated carnitine, sphingosine, ceramide,phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine,phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidicacid.

Additional preferred embodiments of the microparticle-conjugatedbiologically active compounds of the invention also comprise a specificlinker moiety wherein activation of the biologically active compound isachieved by specific cleavage of the linker moiety in a cell, preferablya phagocytic cell, infected with a microorganism, most preferably apathological or disease-causing microorganism.

In a particular embodiment of this aspect of the invention, the specificlinker moiety is a peptide of formula (amino acid)_(n), wherein n is aninteger between 2 and 100, preferably wherein the peptide comprises apolymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, thebiologically-active compound of the invention has a first functionallinker group, and a polar lipid moiety has a second functional linkergroup, and the compound is directly covalently linked to the polar lipidmoiety by a chemical bond between the first and second functional linkergroups. In such embodiments, either the biologically-active compound orthe polar lipid moiety comprises yet another functional linker groupwhich is directly covalently linked to a non-specific cleavable linkermoiety of the invention, which in turn is covalently linked to themicroparticle. In preferred embodiments, each of the functional linkergroups is a hydroxyl group, a primary or secondary amino group, aphosphate group or substituted derivatives thereof or a carboxylic acidgroup. In particular, in such embodiments the polar lipid/biologicallyactive compound conjugate is preferably specifically cleaved in infectedphagocytic mammalian cells. In these embodiments, thebiologically-active compound is an inactive, prodrug state whencovalently linked to the polar lipid, which activity of the biologicallyactive compound in restored or increased after the conjugate has beenbroken.

In specific aspects of the invention provided herein are microparticlescomprising a drug. In preferred embodiments, the drug is an antiviral orantimicrobial drug.

As disclosed herein, the invention comprehends a microparticle and apolar lipid/biologically-active compound conjugate, preferablycomprising a drug, more preferably comprising an antiviral orantimicrobial drug, wherein the conjugate is further covalently linkedto a microparticle, impregnated within a microparticle, or coating amicroparticle, wherein the microparticles are specifically taken up bycells, preferably phagocytic mammalian cells, and wherein the conjugatesof the invention are non-specifically released inside the cell. Inpreferred embodiments, the conjugates of the invention further comprisea specific linker moiety. The specific linker moiety of the conjugatesof the invention preferably releases the drug from the lipid, targetsthe conjugate to a subcellular organelle, incorporate the drug into aviral envelope, or perform other functions to maximize the effectivenessof the drug, wherein the drug is in an inactive or reduced activity formuntil it is specifically released from the conjugate in a cell infectedwith a microorganism, most preferably a pathological or disease-causingmicroorganism. In other preferred embodiments, the biologically-activecompound and the polar lipid are directly linked, preferably covalentlylinked, and the compound is restored from an inactive, prodrug form tothe activity of the biologically-active compound by cleavage of thepolar lipid from the biologically-active compound. In yet otherpreferred embodiments, the biologically-active compound is directlylinked to the microparticle, or impregnated within the microparticle, orcoats the microparticle, and is specifically restored from an inactive,prodrug form to the activity of the biologically-active compound in aphagocytic cell infected by a microorganism, most preferably apathological or disease-causing microorganism.

It will be recognized that heterogenous preparations of saidmicroparticles of the invention, comprising either differentmicroparticles conjugated, impregnated or coated with differentbiologically-active compounds, or one particular species conjugated,impregnated or coated with different biologically-active compounds ofthe invention, explicitly fall within the scope of the inventiondisclosed and claimed herein. Said preparations will be understood tocomprise a multiplicity of the biologically-active compounds of theinvention, preferably provided in an inactive, prodrug form.

The microparticle-drug conjugates of this invention have numerousadvantages. First, the drug-microparticle conjugates are specificallytaken up by cells, particularly phagocytic mammalian cells. Also, drugs,preferably antiviral and antimicrobial drugs comprising thedrug-microparticle conjugates of the invention, are linked to themicroparticle by a cleavable linker moiety that is non-specificallycleaved upon entry into phagocytic cells. More importantly, the drugs,preferably antiviral and antimicrobial drugs, are also preferablyconjugated with a polar lipid, most preferably via a specific linkermoiety. In this form, the drugs have reduced or inhibited biologicalactivity, which activity is restored upon chemical or enzymatic cleavageof the specific linker moiety in appropriate phagocytic cells, forexample, phagocytic cells infected with a microorganisms, preferably apathological or disease-causing microorganism. Third, the drug-polarlipid conjugates of the invention will promote the intracellulartargeting of a variety of potentially useful antiviral or antimicrobialdrugs at pharmicokinetic rates not currently attainable. In this aspect,the range of targeted subcellular organelles is not limited per se by,for example, any particular, limited biological properties of thesubcellular organelle such as the number and type of specific receptormolecules expressed by the organelle. In contrast to traditionalattempts to simply target drugs to specific cells, this method maytarget drugs to specific intracellular organelles and otherintracellular compartments. Fourth, the compositions of matter of theinvention incorporate polar lipid/drug conjugates comprising a variablespecific linker region that may allow pharmacologically-relevant ratesof drug release from polar lipid moieties to be engineered into thecompositions of the invention, thereby increasing their clinicalefficacy and usefulness. Thus, time-dependent drug release and specificdrug release in cells expressing the appropriate degradative enzymes areuniquely available using the microparticle-drug-lipid conjugates of theinvention. Fifth, the conjugates of the invention can be combined withother drug delivery approaches to further increase specificity and totake advantage of useful advances in the art. One example of antiviraltherapy would involve incorporating the conjugates of the invention intothe viral envelope, thereby directly modifying its lipid composition andinfluencing viral infectivity. Finally, the prodrug-microparticleconjugates of the invention specifically encompass prodrugs which arebiologically inactive unless and until pathogen infection-specificchemical or enzymatic cleavage converts such prodrugs into an activedrug form inside a phagocytic mammalian cell.

Thus, the invention also provides a method of killing a microorganisminfecting a mammalian cell. This method comprises contacting an infectedphagocytic, mammalian cells with the compositions of matter of theinvention. The invention also provides a method for treating a microbialinfection in a human wherein the infecting microbe is present inside aphagocytic cell in the human, the method comprising administering atherapeutically effective amount of the compositions of matter of theinvention to the human in a pharmaceutically acceptable carrier. Thus,the invention also provides pharmaceutical compositions comprising thecompositions of matter of the invention in a pharmaceutically acceptablecarrier.

Thus, in a first aspect the invention provides compositions of matterfor targeting biologically active compounds to phagocytic cells. In asecond aspect, the invention provides compositions of matter and methodsfor the specific release of biologically active compounds insidephagocytic cells. The invention in yet a third aspect provides methodsand compositions for intracellular delivery of targeted biologicallyactive compounds to phagocytic cells. The invention also provides fororganelle-specific intracellular targeting of biologically activecompounds, specifically to phagolysosomes and other subcellularstructures including but not limited to the endoplasmic reticulum, theGolgi apparatus, mitochondria and the nucleus. In this aspect of theinvention are also provided compositions and methods for organellespecific intracellular targeting using polar lipid moiety-linkedcompounds. In each of these aspects is provided methods and compoundsfor introducing biologically active compounds into phagocytic mammaliancells wherein the unconjugated compound would not otherwise enter saidphagocytic cell. In this aspect is included the introduction of saidbiologically active compounds in chemical embodiments that would nototherwise enter the cell, for example, as phosphorylated embodiments. Inyet another aspect is provided methods and compositions for the specificcoordinate targeting of more than one biologically active compound to aspecific cell type, that is, phagocytic mammalian cells. In anotheraspect, the invention provides reagents and compositions forintroduction and specific release of antiviral or antimicrobial drugsand other biologically-active compounds into cells infected by apathological microorganism. In a final aspect, the invention providesmethods and reagents for delayed, sustained or controlled intracellularrelease of biologically active compounds conjugated to a micropartricle,or impregnated within a coated, porous microparticle, or coated onto anonporous microparticle, wherein the degradation of either the coating,the cleavable linker, the specific linker moiety, the microparticle orany of these activity control points provides said delayed, sustained orcontrolled intracellular release of the biologically active compound ofthe invention.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthetic scheme put forth in Example 1.

FIG. 2 depicts the synthetic scheme put forth in Example 2.

FIG. 3 depicts the synthetic scheme put forth in Example 3.

FIG. 4 depicts the synthetic scheme put forth in Example 4.

FIG. 5 depicts the synthetic scheme put forth in Example 5.

FIG. 6 depicts the synthetic scheme put forth in Example 6.

FIG. 7 depicts the synthetic scheme put forth in Example 7.

FIG. 8 depicts the synthetic scheme put forth in Example 8.

FIGS. 9A through 9D depict prodrugs tested as in Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions of matter and methods forfacilitating the entry biologically-active compounds into phagocyticcells. For the purposes of this invention, the term “biologically-activecompound” is intended to encompass all naturally-occurring or syntheticcompounds capable of eliciting a biological response or having aneffect, either beneficial or cytotoxic, on biological systems,particularly cells and cellular organelles. These compounds are intendedto include but are not limited to all varieties of drugs, particularlyantimicrobial drugs, defined herein to include antiviral, antibacterial,fungicidal and anti-protozoal, especially anti-plasmodial drugs, as wellas peptides including antimicrobial peptides. Also included in thedefinition of “biologically active compounds” are antineoplastic drugs,particularly methotrexate and 5-fluorouracil and other antineoplasticdrugs.

This invention provides microparticle-linked antiviral and antimicrobialagents for specific cell targeting to phagocytic mammalian cells. Asused herein, phagocytic mammalian cells include but are not limited tomonocytes, macrophages, alveolar macrophages, peritoneal macrophages,Kuppfer cells of the liver, macrophage cells resident in the centralnervous system and the skin, all tissue inflammatory and noninflammatorymacrophages, and phagocytic bone marrow cells.

This invention provides microparticle-linked antimicrobial agentswherein an antiviral or antimicrobial drug is linked to a microparticlevia a cleavable linker moiety. The term “antimicrobial drug” is intendedto encompass any pharmacological agent effective in inhibiting,attenuating, combating or overcoming infection of phagocytic mammaliancells by a microbial pathogen in vivo or in vitro. Antimicrobial drugsas provided as components of the antimicrobial agents of the inventioninclude but are not limited to penicillin and drugs of the penicillinfamily of antimicrobial drugs, including but not limited topenicillin-G, penicillin-V, phenethicillin, ampicillin, amoxacillin,cyclacillin, bacampicillin, hetacillin, cloxacillin, dicloxacillin,methicillin, nafcillin, oxacillin, azlocillin, carbenicillin,mezlocillin, piperacillin, ticaricillin, and imipenim; cephalosporin anddrugs of the cephalosporin family, including but not limited tocefadroxil, cefazolin, caphalexn, cephalothin, cephapirin, cephradine,cefaclor, cefamandole, cefonicid, cefoxin, cefuroxime, ceforamide,cefotetan, cefinetazole, cefoperazone, cefotaxime, ceftizoxime,ceftizone, moxalactam, ceftazidime, and cefixime; aminoglycoside drugsand drugs of the aminoglycoside family, including but not limited tostreptomycin, neomycin, kanamycin, gentamycin, tobramycin, amikacin, andnetilmicin; macrolide and drugs of the macrolide family, exemplified byazithromycin, clarithromycin, roxithromycin, erythromycin, lincomycin,and clindamycin; tetracyclin and drugs of the tetracyclin family, forexample, tetracyclin, oxytetracyclin, democlocyclin, methacyclin,doxycyclin, and minocyclin; quinoline and quinoline-like drugs, such as,for example, naladixic acid, cinoxacin, norfloxacin, ciprofloxacin,ofloxicin, enoxacin, and pefloxacin; antimicrobial peptides, includingbut not limited to polymixin B, colistin, and bacatracin, as well asother antimicrobial peptides such as defensins (Lehrer et al., 1991,Cell 64: 229-230), magainins (Zasloff, 1987, Proc. Natl. Acad. Sci. USA84: 5449-5453), cecropins (Lee et al., 1989, Proc. Natl. Acad. Sci. USA86: 9159-9162 and Boman et al., 1990, Eur. J. Biochem. 201: 23-31), andothers, provided as naturally-occurring or as the result of engineeringto make such peptides resistant to the action of pathogen-specificproteases and other deactivating enzymes; other antimicrobial drugs,including chloramphenicol, vancomycin, rifampicin, metronidazole,ethambutol, pyrazinamide, sulfonamides, isoniazid, and erythromycin.Antiviral drugs, including but not limited to acyclovir, gangcyclovir,azidothymidine, cytidine arabinoside, ribavirin, amantadine,iododeoxyuridine, poscarnet, and trifluridine are also encompassed bythis definition and are expressly included therein.

The invention also provides microparticle-linked antimicrobial agentswherein an antimicrobial agent is a toxin capable of specificcytotoxicity against the microbe, its host cell or both. The term“toxin” is intended to encompass any pharmacological agent capable ofsuch toxicity, including for example ricin from jack bean, diphtheriatoxin, and other naturally-occurring and man-made toxins.

In the antimicrobial agents as provided by this invention, saidantimicrobial drugs are linked to microparticles that are specificallyphagocytized by phagocytic mammalian cells. It is an advantage of thepresent invention that antiviral and antimicrobial drugs arespecifically targeted to phagocytic mammalian cells, including, interalia, monocytes and macrophages as provided further below, by attachmentto the microparticles that are a component of the antimicrobial agentsof the invention. The term “microparticle” as used herein is intended toencompass any particulate bead, sphere, particle or carrier, whetherbiodegradable or nonbiodegradable, comprised of naturally-occurring orsynthetic, organic or inorganic materials, that is specificallyphagocytized by phagocytic mammalian cells.

In one embodiment of the antiviral and antimicrobial agents of theinvention, said microparticle is a porous particle having a defineddegree of porosity and comprised of pores having a defined size range,wherein the antiviral or antimicrobial drugs are impregnated within thepores of the microparticle. In such embodiments, a chemically orenzymatically-degradable coating covers the surface or outside extent ofthe microparticle, wherein the coating is non-specifically degraded,chemically or enzymatically, within a phagocytic cell afterphagocytosis.

In a second embodiment of the invention, the microparticle is either aporous or a nonporous particle. In such embodiments, the surface oroutside extent of the microparticle comprises chemically functionalgroups that form covalent linkages with the antiviral or antimicrobialdrug component of the antimicrobial agents of the invention, preferablyvia a chemically or enzymatically cleavable linker moiety. In suchembodiments, the cleavable linked moiety is non-specifically chemicallyor enzymatically cleaved within a phagocytic cell after phagocytosis.

In a third embodiment of the invention, the microparticle is nonporousand the antiviral or antimicrobial drug is coated on the outside of themicroparticle, the microparticle further coated with a coating materialto control release of the antiviral or antimicrobial drug in aphagocytic cell. In such embodiments, a chemically orenzymatically-degradable coating covers the surface or outside extent ofthe microparticle, wherein the coating is non-specifically degraded,chemically or enzymatically, within a phagocytic cell afterphagocytosis.

The microparticle component of the antiviral or antimicrobial agents ofthe invention include any particulate bead, sphere, particle or carrierhaving a diameter of about 1 to about 1000 nanometers (about 0.001-1μm). The microparticles of the invention are provided comprised ofpolystyrene, cellulose, silica, and various polysaccharides includingdextran, agarose, cellulose and modified, crosslinked and derivatizedembodiments thereof. Specific examples of the microparticles of theinvention include polystyrene, cellulose, dextran crosslinked withepichlorohydrin (Sephadex™, Pharmacia, Uppsala, Sweden), polyacrylamidecrosslinked with bisacrylamide (Biogel™, BioRad, USA), agar, glass beadsand latex beads. Derivatized microparticles include microparticlesderivatized with carboxyalkyl groups such as carboxymethyl, phosphoryland substituted phosphoryl groups, sulfate, sulfhydryl and sulfonylgroups, and amino and substituted amino groups.

In the antimicrobial agents of the invention as provided in one aspect,the microparticles and antiviral and antimicrobial drugs are linked viaa chemically or enzymatically cleavable linker moiety. In another aspectof the antimicrobial agents of the invention, the antiviral andantimicrobial drugs are impregnated within porous microparticles coatedwith a chemically or enzymatically degradable coating. In another aspectof the antimicrobial agents of the invention, antiviral or antimicrobialdrugs coat the external surface of a nonporous microparticle, which isthereafter further coated with a chemically or enzymatically degradablecoating. In all aspects, release of the antiviral or antimicrobial drugis dependent on specific chemical or enzymatic cleavage of the coatingor linker moieties inside phagocytic cells after phagocytosis of theantimicrobial agent.

In certain embodiments, the antimicrobial agents of the invention alsocomprise polar lipids which are chemically conjugated with thebiologically-active compounds of the invention via a specific linkermoiety. As provided by the invention, the biologically active compoundhas a biological activity that is reduced, suppressed, inhibited orablated by formation of the polar lipid conjugate, and that is improved,restored or activated upon chemical or enzymatic cleavage of thespecific linker moiety and liberation of the biologically activecompound from the polar lipid conjugate. Alternatively, the inventionprovides the biologically active compound in an inactive form such as aprodrug, that is specifically activated inside a phagocytic cellinfected with a microorganism, preferably a pathological ordisease-causing microorganism.

Liberation of the biologically active compound from the polar lipidconjugate, or activation of the inactive prodrug, is specificallyachieved inside a phagocytic cell infected with a microorganism,preferably a pathological or disease-causing microorganism, by preparingthe antimicrobial agents of the invention wherein the specific linkermoiety or the prodrug activation is mediated by chemical reaction underphysiological conditions specific for infection of a phagocyticmammalian cell with a particular microorganism, most preferably apathological or disease-causing microorganism. In this embodiment,specific cleavage is due to an chemical linkage which is labile withinthe infected phagocytic cell due to conditions caused by or that resultfrom infection of the phagocytic cell with a particular microbialpathogen. Alternatively, the specific linker moiety or the prodrugactivation is mediated by enzyme action of an enzyme produced by (i.e.,encoded by the microorganism) or induced by (i.e., encoded by the hostphagocytic cell) infection of a phagocytic mammalian cell with aparticular microorganism, most preferably a pathological ordisease-causing microorganism.

Examples of such combinations resulting in specific release of theantiviral or antimicrobial drug component of the antimicrobial agents ofthe invention within infected phagocytic cells include but are notlimited to a urea-based linker for use against a pathogen which producesurease (e.g., Mycobacteria spp. and B. pertussis); a peptide linkercomprised of (AlaAlaAlaAla)_(n), wherein n can be an integer from 1-5,for use against a pathogen that produces the protease oligopeptidase A(e.g., Salmonella spp.); a peptide comprised of from 3 to about 20 aminoacids comprising the sequence -Pro-Xaa-Pro-, where Xaa is any aminoacid, for use against a pathogen that produced proline peptidase (e.g.,Salmonella spp.); peptides comprising the dipeptide MetMet or LeuAla, orpeptides comprising the amino acid sequence GSHLVEAL, HLVRALYL,VEALYLVC, or EALYLVCG, for use against human immunodeficiency virus 1producing a specific protease termed HIV-1 protease; a peptidecomprising the amino acid sequence:-Ala-Xaa-Cys_(Acm)-Tyr-Cys-Arg-Ile-Pro-Ala-Cys_(Acm)-Ile-Ala-Gly-Asp-Arg-Arg-Tyr-Gly-Thr-Cys_(Acm)-Ile-Tyr-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys_(Acm)-Cys_(Acm)-,wherein the microbial pathogen expresses an enzymatic activity thatspecifically disables the endogenous antimicrobial peptide defensin(e.g., Mycobacterium spp. and L. pneumophila), (-Cys_(Acm)-) representcysteine residues having the sidechain sulfur atom protected by covalentlinkage to an acetamidomethyl group (it will be recognized thatembodiments of such peptides having alternative sulfur protecting groupsare also within the scope of the disclosure herein) and Xaa is eitherabsent or Asp; said peptides are also useful as components of themicroparticulate antimicrobial compounds of the invention against apathogen such as Legionelia spp. producing a 39 kDa metalloprotease;hippurate esters that are hydrolyzed by pathogen-specific (e.g., L.pneumophila and Listeria spp.) hydrolase; nicotinic acid amides cleavedby nicotinamidases, pyrazinamides cleaved by pyrazinamidase; ailolactoselinkages cleaved by β-galactosidase; and allantoate linkages cleaved byallantoicase (e.g., Mycobacterium spp.).

In certain specific embodiments, combinations or mixtures of theantimicrobial agents of the invention will comprise the therapeuticpharmaceutical agents of the invention, as provided below. In otherembodiments, said mixtures will include compositions of mattercomprising a microparticle covalently linked to an enzyme having anactivity that recognizes and cleaves the cleavable linker or coatingmoiety of the other antimicrobial agent component of the mixture, saidenzyme-linked microparticles having activity as drug releaseaccelerators. In preferred embodiments, the activity or optimal activityof such enzymatic drug release accelerators will be achieved only insidea phagocytic mammalian cell, for example, in a phagolysosome.

The cleavable linker or coating material comprising the microparticlesof the invention is non-specifically degraded inside a phagocytic cellthat has phagocytized the microparticles. Thus, encompassed within thecleavable linkers and coating materials of the microparticles of theinvention are materials that are chemically or enzymatically degraded ina phagocytic cell using the cellular machinery, enzymes and pathways fordegradation and processing of phagocytized materials. Examples of thetypes of cellular enzymes explicitly recited as being within the scopeof this invention are arylsulfatases, most preferably lysosomalarylsulfatases, as described in Lukatela et al. (1998, Biochemistry 37:3654-3664) and Recksiek et al. (1998, J. Biol. Chem. 273 6096-6103). Forexample, microparticles coated with polymers comprising linear sulfateesters (such as peptides linked tail-to-tail via a sulfate ester linkageto the carboxyl termini of the peptides) are appropriate substrates forarylsulfatases. In another example, chondroitin sulfate coatings areexpected to be non-specifically degraded by lysosomal arylsulfatases.Acid proteases (as described ion Aniento et al., 1997, Electrophoresis18: 2638-2644) are another class of cellular enzymes, substrates ofwhich can be used in the cleavable linkers and coating materials of theinvention. In this embodiment, peptides comprising the sequence(Asp)_(n)(Glu)_(m), (Glu)_(n), or (Asp-Glu)_(n), where 2<n<˜50 and m>1can be used as the cleavable linkers and coating materials (mostpreferably wherein long homopolymeric Asp sequences are avoided) and benon-specifically cleaved in a phagocytic cell. Alternatively, substratesfor lipases, such as cholesterol-fatty acid esters (as described in Duet al., 1998, Gene 208: 285-295) can be used for to comprise thecleavable linkers or coating materials of the microparticles of theinvention.

For the purposes of this invention, the term “non-specific” when usedwith regard to the cleavable linkers or coating materials is intended toindicate that cleavage is not specific for phagocytic cells infectedwith a microorganism, but that the cleavable linker moieties and coatingmaterials are expected to be cleaved in any phagocytic cell. Theparticular mechanisms of cleavage, particularly wherein said mechanismsinvolve enzymatic cleavage, will be characterized by theconventionally-recognized specificity between enzyme and substrate.

In other embodiments, a multiplicity of biologically-active compoundscomprise the microparticle embodiments of the invention.

In specific embodiments of the antimicrobial agents of the invention,said antimicrobial agents are conjugated with a polar lipid targetingmoiety comprised of one or a plurality of polar lipid molecules. Thepolar lipid moiety in such embodiments is covalently linked to eitherthe antiviral or antimicrobial drug via a specific linker moiety asdescribed herein. The polar lipid moiety is linked to the antiviral orantimicrobial drug through an specific linker moiety comprising a firstfunctional linker group and a second functional linker group. The term“polar lipid moiety” as defined herein is intended to mean any polarlipid having an affinity for, or capable of crossing, a biologicalmembrane. Polar lipid moieties comprising said embodiments of theinvention include but are not limited to acyl carnitine, acylatedcarnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidylglycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidylserine, cardiolipin, phosphatidic acid, sphingomyelin and othersphingolipids, as these terms are understood in the art (see, Lehninger,Biochemistry, 2d ed., Chapters 11 & 24, Worth Publishers: New York,1975).

These embodiments of the invention may be further comprised of anspecific linker moiety comprising a first end and a second end, each endof the linker having a functional linking group. For the purposes ofthis invention, the term “specific linker” or “specific linker moiety”is intended to encompass any chemical entity that links abiologically-active compound such as an antiviral or antimicrobial drugand a polar lipid moiety, and having a specificity for a chemical orenzymatic reaction capable of cleaving the specific linker moiety onlywithin a phagocytic cell, preferably a phagocytic mammalian cell,infected with a particular microorganism, most preferably a pathologicalor disease-causing microorganism. Such specific linker moieties aredesigned to facilitate, control, modulate and regulate the release ofthe biologically-active compound at a desired intracellular target site.Such specific linkers also facilitate enzymatic release at certainintracellular sites.

As used herein, the term “linker functional group” is defined as anyfunctional group for covalently linking the polar lipid moiety orbiologically-active agent to the specific linker moiety. This definitionalso includes functional groups comprising a biologically activecompound, the polar lipid, a microparticle or any appropriatecombination thereof.

Linker functional groups can be designated either “weak” or “strong”based on the stability of the covalent bond which the linker functionalgroup will form. The weak functionalities include, but are not limitedto phosphoramide, phosphoester, carbonate, amide, carboxyl-phosphorylanhydride, ester and thioester. The strong functionalities include, butare not limited to ether, thioether, amine, amide and ester. Stronglinker functional groups comprise the functional covalent linkagesbetween the biologically active compounds, the specific linker moietiesand the polar lipids of the conjugates of the invention, wherein theconjugates are chemically stable inside phagocytic cells and are cleavedonly under specific conditions, i.e., infection of the host cell by aparticular microorganism. Enzyme-mediated modes of release will notnecessarily be correlated with bond strength in such embodiments of theinvention; however, strong linkages are intended to minimizeinappropriate release of the biologically active compounds of theconjugates of the invention in cells not infected with a microorganism.Specific linker moieties comprising enzyme active site recognitiongroups, such as linker groups comprising peptides having proteolyticcleavage sites therein, are but one example of the types of specificlinker moieties within the scope of the present invention.

The antimicrobial agents of this invention are useful in inhibiting,attenuating, arresting, combating and overcoming infection of phagocyticmammalian cells with pathogenic microorganisms in vivo and in vitro. Tothis end, the antimicrobial agents of the invention are administered toan animal infected with a pathogenic microorganism acutely orchronically infecting phagocytic mammalian cells. The antimicrobialagents of the invention for this use are administered in a dosage andusing a therapeutic protocol sufficient to have an antimicrobial effectin the phagocytic cells of the animal. Thus, methods of treatingmicrobial infections in a mammal, specifically infections of phagocyticmammalian cells, are provided. Pharmaceutical compositions useful in themethods provided by the invention are also provided.

The following Examples illustrate certain aspects of the above-describedmethod and advantageous results. The following examples are shown by wayof illustration and not by way of limitation.

EXAMPLE 1

An inactive, prodrug form of an antimicrobial agent is prepared byconjugating a non-specifically cleavable peptide (i.e., one that will becleaved in a phagocytic cell) to a derivatized microparticle as follows.An derivatized microparticle comprising unconjugated amino groups isreacted with a proteolytically-inert peptide in which the terminal amineand any of the constituent amino acid sidechain reactive amines arecovered by tert-butoxycarbonyl (t-Boc) protecting groups in the presenceof triphenyl phosphine as described by Kishimoto (1975, Chem. Phys.Lipids 15: 33-36). The peptide/microparticle conjugate is then reactedin the presence of pyridine hydrofluoride as described by Matsuura etal. (1976, J. Chem. Soc. Chem. Comm. xx: 451459) to remove the t-Bocprotecting groups. The peptide/microparticle is then conjugated to thenon-specifically cleavable peptide, in which the terminal amine and anyof the constituent amino acid sidechain reactive amines are covered byt-Boc protecting groups, as described in the presence of triphenylphosphine. After deprotection of reactive amines with pyridinehydrofluoride as described, an inactive, prodrug form of anantimicrobial drug having a reactive carboxylic acid group is conjugatedto a free amino group of themicroparticle/peptide/specifically-cleavable peptide to yield theantimicrobial agent of the invention. This reaction scheme isillustrated in FIG. 1.

EXAMPLE 2

An antiviral compound (HIV1 protease inhibitor; compound 8) isconjugated to sphingosine as follows. Sphingosine is reacted with 1,3bis(trimethylsilyl)urea as described by Verbloom et al. (1981, Synthesis1032: 807-809) to give a trimethylsilyl derivative of sphingosine. Thesphingosine derivative is then conjugated with the antigenically-activepeptide in which the terminal amine and any of the constituent aminoacid sidechain reactive amines are covered by tert-butoxycarbonyl(t-Boc) protecting groups in the presence of diethylazo-dicarboxylate(DEAD) and triphenyl phosphine as described by Kishimoto (1975, Chem.Phys. Lipids 15: 33-36). The sphingosine/peptide conjugate is thenreacted in the presence of pyridine hydrofluoride as described byMatsuura et al. (1976, J. Chem. Soc. Chem. Comm. xx: 451459) to removethe t-Boc protecting group, to yield the antigenically-active peptidecovalently linked to sphingosine through an amide bond. This reactionscheme is illustrated in FIG. 2. Sphingosine/drug conjugates are thenlinked to microparticles as described in Example 1.

EXAMPLE 3

An antiviral compound (compound 8) is conjugated to ceramide via apolyglycine linker as follows and as illustrated in FIG. 3. The aminoterminus of polyglycine is protected by a t-Boc group. Polyglycine isconjugated through its carboxy terminus to ceramide forming an esterlinkage, as described in Anderson et al., ibid. The resulting compoundis then conjugated through the amino terminus of the polyglycineresidue. The amino terminus of Compound 8 is also protected by a t-Bocprotecting group. Conjugation with polyglycyl-sphingosine takes placebetween the amino terminus of the polyglycyl linker moiety and thecarboxy terminus of the HIV-1 protease inhibitor. This reaction iscarried out in the presence of DEAD and triphenyl phosphine as describedin Examples 1 and 2. Following this conjugation, the amino terminus ofthe HIV-1 protease inhibitor residue is deprotected according to themethod of Matsuura et al., ibid. Ceramide/drug conjugates are thenlinked to microparticles as described in Example 1, or used toimpregnate a porous microparticle, or used to coat a non-porousmicroparticle.

EXAMPLE 4

An antiviral compound is prepared wherein ceramide is first conjugatedto a first end of an oligomeric 3-hydroxy propanoic acid linker throughan ester functional group, and wherein AZT is conjugated to a second endof said polyester linker through a phosphodiester bond. First apolyester linker is obtained, having a carboxyl at a first end and atriphenylmethyl group esterified to a second end. This linker isconjugated to ceramide at its first end through an ester functionallinker group according to the method of Anderson et al., ibid. Thiscompound is then conjugated through the second end of the linkercompound to AZT monophosphate by means of a phosphodiester bondaccording to the method of Baer (1955, Can. J. Biochem. Phys. 34: 288).In this antiviral compound, the bond breakage between the linker and thedrug would be slow in the absence of a phosphohydrolase. This reactionscheme is illustrated in FIG. 4. Ceramide/drug conjugates are thenlinked to microparticles as described in Example, 1, or used toimpregnate a porous microparticle, or used to coat a non-porousmicroparticle.

EXAMPLE 5

An antiviral compound wherein phosphatidic acid, phosphatidyl choline,phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol orphosphatidylethanolamine is linked through a phosphoester linkerfunctional group to the antiviral drug azidothymidine (AZT).Phosphatidic acid, phosphatidyl choline, phosphatidyl serine,phosphatidyl inositol, phosphatidyl glycerol or phosphatidylethanolamine is conjugated to AZT according to the method of Salord etal. (1986, Biochim. Biophys. Acta 886: 64-75). This reaction scheme isillustrated in FIG. 5. Phospholipid/drug conjugates are then linked tomicroparticles as described in Example 1, or used to impregnate a porousmicroparticle, or used to coat a non-porous microparticle.

EXAMPLE 6

An antiviral compound is prepared wherein aminohexanoyl sphingosine isconjugated to AZT. Aminohexanoyl sphingosine is conjugated with AZTaccording to the method of Kishimoto (1975, Chem. Phys. Lipid 15:33-36). This reaction scheme is illustrated in FIG. 6 to yieldaminohexanoyl sphingosine conjugated to AZT through a phosphoramidebond. Such conjugates are then linked to microparticles as described inExample 1, or used to impregnate a porous microparticle, or used to coata non-porous microparticle.

EXAMPLE 7

An antiviral compound consisting of ceramide conjugated toAZT-monophosphate is provided. Ceramide is reacted withAZT-monophosphate in the presence of dicyclohexylcarbodiimide asdescribed in Smith and Khorana (1958, J. Amer. Chem. Soc. 80: 1141) toyield ceramide conjugated through a phosphodiester bond toAZT-monophosphate. This reaction scheme is illustrated in FIG. 7. TheAZT/polar lipid conjugate is then linked to microparticles as describedin Example 1, or used to impregnate a porous microparticle, or used tocoat a non-porous microparticle.

EXAMPLE 8

An antiviral compound is prepared wherein ceramide is conjugated throughan ester functional group to a first end of a polyglycine linker, andwherein AZT is conjugated through a phosphoester functional group to asecond end of the polyglycine linker. Ceramide is first conjugatedthrough an ester functional group to a first end of a polyglycine linker(as described in Example 2). The ceramide-polyglycine compound is thenconjugated through a phosphoester bond to a second end of thepolyglycine linker to AZT monophosphate according to the method of Pauland Anderson, ibid. This reaction scheme is illustrated in FIG. 8.Conjugates as prepared herein are then linked to microparticles asdescribed in Example 1, or used to impregnate a porous microparticle, orused to coat a non-porous microparticle.

EXAMPLE 9

The effect of presenting a biologically active compound such as a drugto mammalian cells as a prodrug covalently linked to a polar lipidcarrier moiety was determined as follows. The antifolate drugmethotrexate was conjugated with a variety of polar lipid carriers viaspecific linker moieties having specific reactive functional groups. Arepresentative sample of such compounds is shown in FIGS. 9A through 9C,wherein MC represents Mtx linked to sphingosine via an amide bond to a6-aminohexanoic acid linker, ME₆C represents. Mtx linked to sphingosinevia an ester linkage to a 6-hydroxyhexanoic acid linker, and MSCrepresents Mtx linked to sphingosine via a salicylic acid ester linkageto a 6-aminohexanoic acid linker. Also studied was a conjugate ofazidothymidine linked to sphingosine via an ester linkage to a6-hydroxyhexanoic acid linker (N-AZT-ceramide). The compounds weretested for their growth inhibitory effects on murine NIH 3T3 cellsgrowing in cell culture. About one million such cells per P100 tissueculture plate were grown in DMEM media supplemented with 10% fetal calfserum (GIBCO, Grand island, N.Y.) in the presence or absence of agrowth-inhibitory equivalent of each prodrug. Cell numbers weredetermined after 70 hours growth in the presence or absence of theprodrug. In a second set of experiments was included in the growth mediaan amount of a brain homogenate containing an enzymatically-activeesterase.

The results from these experiments are shown in Table I. As can be seenfrom these data, the MC prodrug had no effect on the growth and survivalof the cells. This result did not change upon co-incubation with theesterase-containing brain extract, which was expected due to the natureof the drug/linker linkage (an amide bond). A different result wasobtained with the ME₆C conjugate. The prodrug was ineffective ininhibiting cell growth or survival in the absence of brain extract. Uponaddition of the brain extract, a significant increase in Mtxcytotoxicity was observed. This is consistent with cleavage of the esterlinkage by the brain extract-derived esterase. A similar result wasobtained with the MCS conjugate, indicating that the brain extractesterase activity was capable of cleaving the salicylic acid ester.

Table II shows the results of drug uptake studies performed with theprodrug N-AZT-ceramide. Antiviral amounts of the prodrug conjugate wereadded to NIH 3T3 cell cultures, and the antiviral activity of theprodrug was found to be equivalent to the activity of free AZT. Inaddition, upon removal of the prodrug, intracellular retention ofprodrug was found to be up to 15-fold higher than free AZT (Table II)over a 23 h period.

These results indicate that for Mtx-containing conjugates, the free drugmust be released from the prodrug for biological activity. These resultssuggest that specific release of this drug, and perhaps others, can beachieved using cleavable linker moieties that are specifically cleavedonly in pathogen-infected cells. TABLE I Sample¹ # cells/plate² Sample³# cells/plate⁴ Control/FBS 7.8 × 10⁶ Control/FBS   13 × 10⁶ ME₆C/FBS 6.5× 10⁶ MSC/FBS  2.1 × 10⁶ ME₆C/brain 2.7 × 10⁶ MSC/brain 0.51 × 10⁶Mtx/FBS 0.16 × 10⁶  Mtx/FBS 0.13 × 10⁶ Mtx/brain 0.09 × 10⁶  Mtx/brain0.06 × 10⁶ Control/brain N.D. Control/brain  6.2 × 10⁶¹= cells incubated with drug/FBS or drug/brain extract for 1 hour at 37°C.²= cell growth and survival determined 70 hours after drug addition³= cells incubated with drug/FBS or drug/brain extract for 2 hours at37° C.⁴= cell growth and survival determined 72 hours after drug addition

Time¹ AZT² N-AZT-Ceremide²  0 hr. 6.49 8.45 23 hr. 0.55 7.78¹= time between the end of drug treatment and assay for intracellulardrug concentration²= nM/10⁶cells

EXAMPLE 10

Antimicrobial agents of the invention are used as follows. Theantimicrobial agent or a negative control (saline) are administered toan animal infected with a microbial pathogen using both optimal andsuboptimal dosages and the most appropriate route of administration.After an optimal time period (determined from the nature of theinfection), phagocytic cells are collected from the animal and testedfor infection with the microbial pathogen. Phagocytic cells fromperipheral blood are isolated using conventional methods (Ficoll-Hypaquedensity gradient centrifugation) and tested for the presence ofinfectious microbial pathogens using conventional immunological,microbiological and biochemical testing protocols (see Laboratory TestHandbook, Jacobs et al., eds., Lexi-Comp, Inc: Cleveland, Ohio, 1994;Clinical Laboratory Medicine, McClatchey, ed., Williams & Wiklins:Baltimore, Md., 1994; Clinical Diagnosis and Management by Laboratory,18th Ed., J. B. Henry, ed., W. B. Saunders: Philadelphia, 1991).

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

1. A composition of matter comprising a prodrug of a biologically-active compound, a nonporous microparticle, and an organic coating material, wherein the prodrug comprises a biologically active compound, a polar lipid moiety comprised of one or a plurality of polar lipid molecules and a specific linker moiety, wherein the specific linker moiety is covalently linked to both the biologically active compound and the polar lipid moiety, wherein the nonporous microparticle is first coated with the prodrug and said coated microparticle is further coated with the organic coating moiety, and wherein the organic coating material is nonspecifically degraded inside a phagocytic mammalian cell infected with a microorganism to allow release of the prodrug within the infected cell, and wherein the specific linker moiety is specifically cleaved in a phagocytic mammalian cell infected with a microorganism and the prodrug is specifically activated thereby.
 2. The composition of matter of claim 1 wherein the biologically-active compound is a peptide.
 3. The composition of matter of claim 2 wherein the peptide is an antiviral peptide or an antimicrobial peptide.
 4. The composition of matter of claim 1 wherein the prodrug is a prodrug of an antiviral drug or an antimicrobial drug.
 5. The composition of matter of claim 1 wherein the prodrug is a toxin.
 6. A composition of matter according to claim 1 wherein the organic coating material is chemically degraded inside a mammalian phagocytic cell infected with a microorganism.
 7. A composition of matter according to claim 1 wherein the organic coating material is a substrate for a protein having an enzymatic activity, said protein being produced in a mammalian cell infected with a microorganism.
 8. The composition of matter of claim 7 wherein the organic coating material is a substrate for a protein produced by the infected mammalian cell.
 9. A composition of matter according to claim 1 wherein the polar lipid is acyl carnitine, acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.
 10. A method of killing a virus or microorganism infecting a mammalian cell, the method comprising contacting said cell with the composition of claim
 3. 11. A method of killing a virus or microorganism infecting a mammalian cell, the method comprising contacting said cell with the composition of claim
 5. 12. A method for treating a viral infection in a human wherein the infecting virus is present inside a phagocytic cell in the human, the method comprising administering a therapeutically effective amount of the composition of claim 3 to the human in a pharmaceutically acceptable carrier.
 13. A method for treating a microbial or viral infection in a human wherein the infecting microbe is present inside a phagocytic cell in the human, the method comprising administering a therapeutically effective amount of the composition of claim 5 to the human in a pharmaceutically acceptable carrier.
 14. A pharmaceutical composition comprising the composition of matter of claim 1 in a pharmaceutically acceptable carrier. 